This application is related to U.S. patent application Ser. No. 08/989,560 to Jeffrey et al. filed Dec. 12, 1997, and U.S. patent application Ser. No. 09/113,929 to Maresch et al. (now U.S. Pat. No. 5,912,115), the subject matter of each being incorporated herein by reference.
The presence of microbial contamination in clinical specimens is conventionally determined by culturing the specimens in the presence of nutrients and detecting microbial activity through changes in the specimen or in the atmosphere over the specimen after a period of time. For example, in the U.S. Pat. No. 4,182,656 to Ahnell et al., the sample is placed in a container with a culture medium comprising a carbon 13 labeled fermentable substrate. After sealing the container and subjecting the specimen to conditions conducive to biological activity, the ratio of carbon 13 to carbon 12 in the gaseous atmosphere over the specimen is determined and compared with the initial ratio. In U.S. Pat. No. 4,152,213, a method is claimed by which the presence of oxygen consuming bacteria in a specimen is determined in a sealed container by detecting a reduction in the amount of oxygen in the atmosphere over the specimen through monitoring the pressure of gas in the container. U.S. Pat. No. 4,073,691 provides a method for determining the presence of biologically active agents, including bacteria, in a sealed container containing a culture medium by measuring changes in the character of the gaseous atmosphere over the specimen after a period of time.
A method for non-invasive detection is taught by Calandra et al., U.S. Pat. No. 5,094,955, where a device is disclosed for detecting the presence of microorganisms in clinical specimens, such as blood or other body fluids, and in non-clinical specimens, by culturing the specimens with a sterile liquid growth medium in a transparent sealed container. The presence of microorganisms is determined by detecting or measuring changes in the pH of the specimen or the production of carbon dioxide within the specimen using a sensor affixed to the interior surface of the container or to the sealing means used to seal the container. In Calandra et al., microorganisms can be detected in the presence of interfering material, such as large concentrations of red blood cells, through non-radiometric and non-invasive means.
One disadvantage of the detection system of Calandra et al., is that the time required for detecting the presence of microorganisms is related to the number of microorganisms within the sample. Also, the growth medium for the microorganisms is a liquid, such that the container must usually be agitated during incubation. This involves the additional expense in making the incubation equipment, as well as an increase in the likelihood of a mechanical breakdown. Also, such a system allows for the determination of the presence of microorganisms, but does not allow for enumeration. Furthermore, it is often the case that after detection of microorganisms, it is desired to identify the microorganisms and/or determine their susceptibility to various antibiotics. In a Calandra-type system, it would be necessary to plate out the microorganisms from the liquid culture medium to assure isolation of mixed species before performing susceptibility or identification tests. This involves additional time (time that is not always available if the patient is very ill). Also, a Calandra-type system could not serve the additional functions of reading/imaging plates for antibiotic susceptibility and/or microbial identification.
There are also known methods for detecting microorganisms where a sample is plated onto a gel (agar) plate. In such methods, a sample is swabbed or xe2x80x9cstreakedxe2x80x9d across the gel plate and microorganism growth is determined by viewing the plate to see if any growth occurs where the sample was swabbed on the gel. Though this type of detection is desirable for its surface colonies (which can be immediately tested for antibiotic susceptibility and/or microbial identification), it is undesirable in that it can not handle the large sample volumes required by many procedures such as blood culture.
Some conventional systems that facilitate simultaneous microbial culture and isolation from bulk liquid involve the use of dehydrated granular gelling medium, or a liquid gelling component that forms a gel after the microorganisms are introduced. In either case, the microorganisms are trapped throughout the medium, not just on the surface. This complicates the harvest of microorganisms for further testing.
Surface culture of microorganisms from a liquid sample can be done on a standard semisolid media such as agar. However, such media are too rigid to swell substantially, and can only absorb a volume of liquid that is typically less than 5% of the starting gel volume. Filtration methods can capture microorganisms from larger volumes of liquid, but have a number of disadvantages, which include increased hands-on time, high cost of materials, risk of contamination, and difficulty with particulate containing samples.
In the present invention, a xe2x80x9cbulkxe2x80x9d fluid sample, possibly containing microorganisms, is poured or otherwise applied to the surface of a gel matrix. The fluid in the sample is absorbed by the gel, yet microorganisms are retained at the surface. After incubation, mutually isolated microorganism colonies are readily accessible on the surface of the medium for harvest and further testing. This provides the advantage that microorganism culture and isolation from a bulk fluid, previously done as a two step process, can be accomplished in a single step, cutting one or more days from the time required to attain a clinically relevant result. Another advantage is that, with localized microorganism growth, the metabolic changes in the culture environment caused by microorganism growth are also localized, which makes detection of these changes, and hence the microorganisms themselves, easier and faster.
The gel matrix of the present invention is preferably composed of a polymeric material that, by nature and/or fabrication technique, offers a unique set of properties. The gel matrix is sufficiently absorptive to draw in the excess fluid from the sample, but with a small enough pore size to filter out or ensnare microorganisms at the surface. The fluid in the sample is absorbed by the gel with sufficient rapidity that, on a time scale typically ranging from a few minutes to a few hours, multiplying microbes form discrete colonies rather than spreading across the surface of the medium.
While the range of polymers (in the preferred embodiment) appropriate for this invention that are useful as starting materials is practically infinite, the physical properties useful to the present invention are well defined. The polymer must 1) form a gel or highly viscous solution wherein bulk flow of fluid is arrested under the test conditions, 2) absorb fluid from an aqueous solution or suspension without losing cohesion, 3) retain microorganisms to be cultured on or near the surface and largely immobilized, and 4) permit the growth of the microorganisms of interest. Gel matrices of the present invention that are fabricated from a variety of polymeric materials have been shown to have all of these properties, and will isolate and grow microorganisms from sample volumes several times greater, per unit area, than is possible with agar plates.
The device of the present invention comprises a container and within the container an immobilization layer made of an interconnected network of polymer chains, wherein interstitial spaces between the interconnected network of polymer chains are of a size on average less than an average size of microorganisms to be cultured, such that substantially all (or all) of the microorganisms in a sample during culturing are immobilized on the surface of said immobilization layer. A volume of at least 0.04 ml per each square centimeter of surface area of the immobilization layer can be added and absorbed by the immobilization layer, while maintaining microorganism colonies on the surface of the immobilization layer. The bulk fluid sample can be whole blood (or some fraction), other body fluid, manufacturing fluid, food sample, or the like.